Application of rice OsPBL8 gene in enhancing resistance of rice to Magnaporthe oryzae

Abstract

The application relates to the field of biotechnology, in particular to application of a rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae. The rice OsPBL8 gene is a rice endogenous gene involved in processes such as regulation of plant growth and development, non-biological stress response and hormone signal pathway. The application provides application of the OsPBL8 gene in the field of disease resistance. By gene editing, the OsPBL8 gene is overexpressed, the expression amount of the plant intracellular receptor-like cytoplasmic kinase OsPBL8 is increased, a plurality of signal pathways are regulated, and the disease resistance of the plant is synergistically improved. By regulating the key hub node of the targeted immune signal transduction of the plant, more persistent and broad-spectrum disease resistance can be provided, the disease resistance has good universality, the disease resistance and growth can be balanced, the disease resistance has good practicability, and the disease resistance of various rice varieties can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae. BACKGROUND

[0002] Magnaporthe oryzae is a disease that occurs on rice caused by Magnaporthe oryzae, which can occur throughout the growth period of rice, and can harm seedlings, leaves, ears, and joints, respectively known as seedling blight, leaf blight, ear blight, and joint blight. Magnaporthe oryzae is widely distributed and has a great impact on rice yield. In order to prevent and control Magnaporthe oryzae, current methods mainly include cultivating high-quality seedlings, managing water and fertilizer, strengthening field management, implementing control measures, and using chemical agents.

[0003] In order to reduce the adverse effects of Magnaporthe oryzae on rice yield, researchers in the field use biotechnology to improve the varieties of rice. For example, Chinese patent CN112779271A provides rice gene OsFd2 and its application in rice resistance to Magnaporthe oryzae. However, the current transgenic rice still has low resistance to Magnaporthe oryzae, so further research on genes affecting the resistance of rice to Magnaporthe oryzae is still needed. SUMMARY

[0004] The purpose of the present application is to provide a research direction for improving the resistance of rice to Magnaporthe oryzae.

[0005] To achieve the above purpose, the present application provides the following technical solutions:

[0006] Application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae.

[0007] Optionally, the nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, its coding sequence is SEQ ID No: 2, and the amino acid sequence of the encoded receptor-like cytoplasmic kinase is SEQ ID No: 3.

[0008] Optionally, the OsPBL8 gene sequence is transferred into the initial rice plant to form the transgenic rice with overexpression of the OsPBL8 gene.

[0009] Optionally, the transgenic rice is formed by Agrobacterium-mediated transformation method using Agrobacterium strain EHA105 loaded with the OsPBL8 gene to transfect the initial rice plant.

[0010] Optionally, the variety of the initial rice is Nipponbare.

[0011] Optionally, the ability of the transgenic rice to burst reactive oxygen under the induction of chitin is stronger than the ability of the initial rice to burst reactive oxygen under the induction of chitin.

[0012] Optionally, the stem base of the transgenic rice or the initial rice is soaked in a treatment liquid containing the chitin.

[0013] Optionally, the concentration of the chitin in the treatment liquid is any value ranging from 3 μg / mL to 5 μg / mL.

[0014] Optionally, the overexpression of the OsPBL8 gene promotes the expression of resistance genes phenylalanine ammonia lyase (PAL) and WRKY transcription factor 45 (WRKY45).

[0015] The present application has the beneficial effect that by regulating the key pivotal nodes of targeted immune signaling of plants through transgenic technology, more persistent broad-spectrum disease resistance can be provided, and the disease resistance and growth can be balanced, and the practicality is good, which is helpful to improve the disease resistance of various rice varieties.

[0016] The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, and the content of the specification can be implemented. The following is a detailed description of the preferred embodiments of the present application with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is an agarose gel electrophoresis diagram of DNA of multiple rice plants in embodiment one of the present application;

[0018] Figure 2 Figure 2 is an expression amount analysis diagram of the OsPBL8 gene of multiple rice plants in embodiment one of the present application;

[0019] Figure 3 Figure 3 is a phenotype of the leaves of multiple rice plants inoculated with Magnaporthe grisea spores in embodiment one of the present application;

[0020] Figure 4 Figure 4 is a relative biomass analysis diagram of the leaves of multiple rice plants inoculated with Magnaporthe grisea spores in embodiment one of the present application;

[0021] Figure 5 Figure 5 is an active oxygen release amount analysis diagram of the base of multiple rice plants treated with chitin in embodiment one of the present application;

[0022] Figure 6 Figure 6 is an expression analysis diagram of resistance genes in the leaves of multiple rice plants in embodiment one of the present application. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described clearly and completely in connection with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0024] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0025] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0026] In addition, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0027] Application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae.

[0028] The rice OsPBL8 gene is an endogenous gene of rice involved in plant growth and development, non-biological stress response, and regulation of hormone signal pathways. The present application provides the application of OsPBL8 gene in the field of rice blast resistance. By gene editing to overexpress OsPBL8 gene, the expression amount of the plant's receptor-like cytoplasmic kinase OsPBL8 is increased, thereby regulating multiple signal pathways and synergistically realizing the plant's disease resistance.

[0029] By regulating the key hub node of targeted immune signal transduction of the plant through transgenic technology, more persistent and broad-spectrum disease resistance can be provided, which has good universality, balances disease resistance and growth, has good practicality, and helps to improve the disease resistance of various rice varieties.

[0030] The optimization of the traits of agricultural products by transgenic technology is usually based on gene introduction or replacement, and usually relies on the specific recognition mechanism of "gene to gene", and the vertical resistance of the plant is improved based on a single gene. In this application, the function of the endogenous gene is optimized, the improvement direction is more accurate and sustainable, the network regulation of the targeted immune signal transduction in the plant is realized, the horizontal resistance is realized through signal integration, thereby providing more persistent and broad-spectrum disease resistance, breaking through the thinking limitation of traditional disease-resistant genes, and providing a new direction for crop disease-resistant breeding.

[0031] In some embodiments, the nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, the coding sequence thereof is SEQ ID No: 2, and the amino acid sequence of the receptor-like cytoplasmic kinase encoded thereby is SEQ ID No: 3.

[0032] In some embodiments, the OsPBL8 gene sequence is introduced into an initial rice plant to form a transgenic rice with overexpression of the OsPBL8 gene.

[0033] In some embodiments, the transgenic rice is formed by a method of Agrobacterium-mediated transformation of an initial rice plant by an Agrobacterium strain EHA105 loaded with the OsPBL8 gene.

[0034] In some embodiments, the transgenic rice has a stronger ability to burst reactive oxygen under the induction of chitin than the initial rice.

[0035] In some embodiments, the stem base of the transgenic rice or the initial rice is soaked in a treatment liquid containing chitin.

[0036] In some embodiments, the concentration of chitin in the treatment liquid is any value ranging from 3 μg / mL to 5 μg / mL, for example, any value of 3 μg / mL, 4 μg / mL and 5 μg / mL.

[0037] In some embodiments, the overexpression of the OsPBL8 gene promotes the expression of the resistance genes phenylalanine ammonia lyase (PAL) gene and WRKY transcription factor 45 (WRKY45).

[0038] For details, please refer to the following examples.

[0039] Example 1:

[0040] The rice OsPBL8 gene was inserted into the initial rice genome to obtain transgenic rice, and the OsPBL8 gene was overexpressed. The initial rice used in this embodiment is japonica rice, and the variety is Nipponbare. The nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, the coding sequence (CDS) is SEQ ID No: 2, and the amino acid sequence of the encoded receptor-like cytoplasmic kinase is SEQ ID No: 3. The specific operation method for preparing transgenic rice is as follows:

[0041] According to the sequence of the rice OsPBL8 gene, primers OsPBL8-F and OsPBL8-R were designed to amplify the complete open reading frame (ORF) thereof, wherein the nucleotide sequence of OsPBL8-F is SEQ ID No: 4, and the nucleotide sequence of OsPBL8-R is SEQ ID No: 5. OsPBL8-F and OsPBL8-R were both prepared into a solution with a concentration in the range of 10 pmol / L to 15 pmol / L.

[0042] The total RNA of the Nipponbare variety rice plant was used as a template to reverse transcribe the first strand cDNA, and the first strand cDNA was used as an amplification template, and OsPBL8-F and OsPBL8-R were used as amplification primers for amplification. The polymerase required for amplification was a premixed DNA polymerase kit of the PrimeSTAR brand of Takara (Dalian) Biotechnology Co., Ltd., which includes 5×PrimeSTAR GXL Buffer, dNTP Mixture, and PrimeSTAR GXL DNA Polymerase. The specific amplification system is shown in Table 1 below.

[0043]

[0044] The reaction conditions of the amplification system are shown in Table 2 below.

[0045]

[0046] After amplification, agarose gel electrophoresis was performed and the gel was cut to recover the amplification product with an agarose gel recovery kit purchased from Omega Bio-Tek to obtain a higher-purity OsPBL8 gene fragment. The OsPBL8 gene fragment was connected to a special vector for blunt-end cloning provided in a blunt-end cloning kit purchased from TransGen Biotech Co., Ltd. of Beijing, and the connection product was transferred to E. coli DH5a competent cells purchased from Beijing Tian Gen Biotechnology Co., Ltd. to obtain a modified bacterial liquid.

[0047] The modified bacterial liquid was spread on Luria-Bertani (LB) agar plate medium containing 100 mg / mL ampicillin (Amp), and after 12 h of culture at 37°C, colonies were picked and the length of the inserted fragment in the bacterial body was confirmed to be consistent with the expected length by colony polymerase chain reaction (PCR). The bacterial body was sent to Nanjing Kings-R Biotech Co., Ltd. for sequencing, and the sequence determination results showed that the bacterial body was a nucleotide sequence including the OsPBL8 gene in the gene sequence and could encode the protein encoded by the OsPBL8 gene. The plasmid in the bacterial body with correct sequencing was extracted, and the obtained plasmid was named pEASY-Blunt-OsPBL8.

[0048] The pCAMBIA1300.1 vector purchased was single-enzyme cut with self-restriction endonuclease Sac I purchased from Thermo Fisher Scientific, and the enzyme cutting buffer for pCAMBIA1300.1 was 10 times enzyme cutting buffer (10×Buffer), which was purchased together with pCAMBIA1300.1, and the enzyme cutting system is shown in Table 3 below.

[0049]

[0050] After 1 h of enzyme digestion at 37 °C, agarose gel electrophoresis was performed and the gel was cut. The enzyme-digested vector was recovered using an agarose gel recovery kit from Omega Bio-Tek and was reserved for later use. Nanjing Kings-R Bio-technology Co., Ltd. was commissioned to synthesize a DNA sequence of double copies of the Cauliflower mosaic virus (CAMV) 35S promoter, and a DNA fragment with the nucleotide sequence of SEQ ID No: 6 was added at the 5' end, and a DNA fragment with the nucleotide sequence of SEQ ID No: 7 was added at the 3' end. The artificially synthesized DNA fragment was named 2x35s. The 2x35s fragment was homologously recombined into the vector recovered from the pCAMBIA1300.1 vector after enzyme digestion using an In-Fusion brand recombination kit from Takara Bio Inc. The recombination product was transformed into E. coli DH5a competent cells purchased from Beijing Tiangen Bio-technology Co., Ltd. using the heat shock method. All the transformed cells were uniformly spread on Luria-Bertani solid medium containing 50 mg / L kanamycin, and after 16 h of culture at 37 °C, colonies were picked and sent to Nanjing Kings-R Bio-technology Co., Ltd. for sequencing. The plasmid extracted from the colony with correct sequencing was named pCAMBIA1300-2x35s.

[0051] The prepared pCAMBIA1300-2x35s plasmid was single-digested with self-restriction endonuclease BamHI purchased from ThermoFisher Scientific, and the enzyme digestion system is shown in Table 4 below.

[0052]

[0053] After 1 h of enzyme digestion at 37 °C, agarose gel electrophoresis was performed and the gel was cut. The enzyme-digested vector was recovered using an agarose gel recovery kit from Omega Bio-Tek and was reserved for later use.

[0054] The pEASY-Blunt-OsPBL8 plasmid prepared in the above experiment was used as a template, and the corresponding primers OsPBL8-BamHI-F and OsPBL8-BamHI-R were used for amplification. The nucleotide sequence of OsPBL8-BamHI-F is SEQ ID No: 8, and the nucleotide sequence of OsPBL8-BamHI-R is SEQ ID No: 9. After the amplification product was recovered and purified, the purified amplification product was cloned into the product recovered after BamHI enzyme digestion of pCAMBIA1300-2×35s using the recombination kit of the In-Fusion brand of Takara Co., Ltd., and the recombinant product was transformed into the E. coli DH5α competent cells purchased from Beijing Tiangen Biotech Co., Ltd. by heat shock method. The whole bacterial liquid obtained by transformation was uniformly coated on Luria-Bertani solid medium containing 50 mg / L kanamycin. After 37°C culture for 16 h, colonies were picked and sent to Nanjing Kingsriver Biotech Co., Ltd. for sequencing. The plasmid extracted from the colony with correct sequencing was named pCAMBIA1300-2×35s-OsPBL8.

[0055] In this embodiment, pCAMBIA1300-2×35s-OsPBL8 was first transformed into Agrobacterium, and then OsPBL8 gene sequence was transformed into rice plants by Agrobacterium-mediated transformation. The Agrobacterium used in this embodiment was Agrobacterium strain EHA105 purchased from Shanghai Weidi Biotechnology Co., Ltd. pCAMBIA1300-2×35s-OsPBL8 was transformed into the strain by liquid nitrogen freeze-thaw method, thereby obtaining the transformed strain. The specific method includes:

[0056] The EHA105 competent cells were thawed in ice bath, at least 100 ng of plasmid was added, and then gently mixed, ice bathed for 5 min, quickly frozen in liquid nitrogen for 5 min, heat shocked at 37°C for 5 min, and then quickly placed on ice for 1 min to 2 min after completion. 800 μL of antibiotic-free Luria-Bertani medium was added to the system, and recovery was performed by oscillation at 200 rpm in a 28°C shaker for 3.5 h, and then centrifuged at 4000 rpm for 3 min. The surface medium was aspirated with a pipette, and the remaining bacterial liquid was mixed. The bacterial liquid was smeared on solid Luria-Bertani medium containing 100 mg / mL kanamycin and 100 mg / mL rifampicin, and incubated at 28°C for 30 h to 48 h. Colonies were picked for colony polymerase chain reaction, and the obtained positive clone was named EHA105:pCAMBIA1300-2×35s-OsPBL8, and stored at 4°C for standby.

[0057] The EHA105:pCAMBIA1300-2x35s-OsPBL8 was used to transform rice plants of Nipponbare variety by a conventional Agrobacterium-mediated method, and 7 transgenic plants of EHA105:pCAMBIA1300-2x35s-OsPBL8 were obtained in T0 generation.

[0058] The genomic DNA of the above 7 single plants was used as a template to amplify and detect the specific fragment in the transgenic genetic transformation marker gene, i.e., the hygromycin phosphotransferase gene, by using primers hyg283-F and hyg283-R. The nucleotide sequence of hyg283-F is SEQ ID No: 10, and the nucleotide sequence of hyg283-R is SEQ ID No: 11. The 2x Taq DNA Polymerase Mix purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd. was used for amplification. The specific amplification system is shown in Table 5 below.

[0059]

[0060] The genomic DNA of EHA105:pCAMBIA1300-2x35s-OsPBL8 was used as a template to form a positive control group, and water was used as a template to form a negative control group. The DNA molecular weight marker (DNA Marker) was labeled as M, the positive control group was labeled as +, the negative control group was labeled as -, and the experimental systems for detecting the genomic DNA of the 7 single plants were labeled with numbers 1 to 7. The reaction conditions of the amplification system are shown in Table 6 below.

[0061]

[0062] The amplification products were separated by agarose gel electrophoresis, photographed by a gel imaging instrument, and the results were recorded.

[0063] See Figure 1 It can be seen that the electrophoretograms of the DNA of the 7 transgenic plants of EHA105:pCAMBIA1300-2x35s-OsPBL8 are similar to the electrophoretogram of the positive control template, indicating that the target product is amplified.

[0064] The plant corresponding to the DNA corresponding to the target product and numbered 1 was named OsPBL8-OE1, the plant corresponding to the DNA corresponding to the target product and numbered 6 was named OsPBL8-OE6, and the Nipponbare variety rice plant without transgenic treatment was named NIP.

[0065] The whole plants of OsPBL8-OE1, OsPBL8-OE6 and NIP were taken, and total RNA was extracted, and the expression of OsPBL8 gene in the tissue samples was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). In the real-time fluorescent quantitative polymerase chain reaction, the primers used for reverse transcription were RT-OsPBL8-F and RT-OsPBL8-R, wherein the nucleotide sequence of RT-OsPBL8-F was SEQ ID No: 12, and the nucleotide sequence of RT-OsPBL8-R was SEQ ID No: 13. Please refer to Figure 2 It can be seen that the expression amount of OsPBL8 gene in OsPBL8-OE1 and OsPBL8-OE6 is significantly up-regulated compared with NIP, which indicates that the overexpression of OsPBL8 gene occurs in OsPBL8-OE1 and OsPBL8-OE6.

[0066] OsPBL8-OE1, OsPBL8-OE6 and NIP were inoculated with rice blast, and the inoculation method included:

[0067] The rice blast spores were inoculated on complete (CM) medium for activation, and cultured at 28°C for 12 days, and treated with 12h light and 12h dark every 24h. The medium was washed with 0.05% Tween 20 sterilized water, and the spore solution was washed off with a glass rod, filtered with a 40μm filter membrane, and the spore concentration was detected under a microscope, and finally adjusted to 1×10 5 The spore suspension was uniformly sprayed on the surface of rice leaves, and placed in a 28°C incubator, and after 24h dark treatment, treated with 12h light and 12h dark cycle, and observed the phenotype after 7 days of culture.

[0068] Please refer to Figure 3 It can be seen that the lesion length of OsPBL8-OE6 after inoculation with rice blast spores is about 0.3cm, and the lesion length of OsPBL8-OE1 after inoculation with rice blast spores is about 0.2cm, and the lesion length of NIP after inoculation with rice blast spores is significantly larger, which indicates that the insertion of OsPBL8 gene helps to improve the ability of rice to resist rice blast.

[0069] Seven days after spraying Magnaporthe grisea spores, leaf samples of OsPBL8-OE1, OsPBL8-OE6 and NIP were taken to extract DNA, and the contents of rice internal reference gene UBQ and Magnaporthe grisea internal reference gene MoPot in the DNA were detected by real-time fluorescent quantitative polymerase chain reaction, so as to obtain the biomass of Magnaporthe grisea in OsPBL8-OE1, OsPBL8-OE6 and NIP relative to the leaf tissue, i.e. the relative biomass. When detecting the content of UBQ gene by real-time fluorescent quantitative polymerase chain reaction, the primers used for reverse transcription were UBQ-RT-F and UBQ-RT-R, the nucleotide sequence of UBQ-RT-F was SEQ ID No: 14, and the nucleotide sequence of UBQ-RT-R was SEQ ID No: 15. When detecting the content of MoPot gene by real-time fluorescent quantitative polymerase chain reaction, the primers used for reverse transcription were MoPot-RT-F and MoPot-RT-R, the nucleotide sequence of MoPot-RT-F was SEQ ID No: 16, and the nucleotide sequence of MoPot-RT-R was SEQ ID No: 17. The experimental results are shown in Table 2. Figure 4 It can be seen that the relative biomass of Magnaporthe grisea in the OsPBL8 gene overexpression lines OsPBL8-OE1 and OsPBL8-OE6 is significantly lower than that of the control group NIP, indicating that the development of rice blast is inhibited.

[0070] The rice seeds obtained from culturing OsPBL8-OE1, OsPBL8-OE6 and NIP were peeled and sterilized, and then were placed in Hoggland nutrient solution in a glass tube, and were cultured aseptically for 7 days to obtain multiple rice seedlings. 150 μL of double distilled water was added to each well of a black 96-well enzyme-coated plate, and the rice seedlings with uniform size and stem thickness were selected, and the stem base was cut to obtain a small piece with a length of about 1 mm, and the small piece samples obtained by cutting were placed in the small holes of the black enzyme-coated plate, 5 small pieces per hole. When cutting, the white stem with a length of about 2 mm near the seed was removed, and then the stem base tissue was cut. The black enzyme-coated plate was placed at room temperature in the dark overnight, and the room temperature was detected to be 25°C. On the second day, a chitin mother liquor with a concentration of 2 mg / mL was prepared, and 10 mL of reaction solution was prepared therefrom. The preparation method of the reaction solution includes: adding 20 μL of horseradish peroxidase (HRP) solution, 20 μL of chemiluminescent probe L-012 solution and 20 μL of chitin mother liquor into a test tube, and adding double distilled water to make up to 10 mL. The horseradish peroxidase solution is a laboratory-prepared solution, which is prepared by adding 15 mg of horseradish peroxidase powder to 1 mL of sterile water, and is stored in a -20°C refrigerator. The chemiluminescent probe L-012 solution is a laboratory-prepared solution, which is prepared by adding 15 mg of chemiluminescent probe L-012 powder to 1 mL of sterile water, and is stored in a -20°C refrigerator. The black enzyme-coated plate placed in the dark overnight was taken out, the liquid in each hole was sucked out with a pipette and discarded, and 100 μL of reaction solution or double distilled water was added, respectively, to obtain six groups of samples treated with reaction solution or water, wherein the sample obtained by treating NIP with chitin is labeled as group one, the sample obtained by treating OsPBL8-OE1 with chitin is labeled as group two, the sample obtained by treating OsPBL8-OE6 with chitin is labeled as group three, the sample obtained by treating NIP with water is labeled as group four, the sample obtained by treating OsPBL8-OE1 with water is labeled as group five, and the sample obtained by treating OsPBL8-OE6 with water is labeled as group six. The enzyme-coated plate without cover was placed in an enzyme marker, and each hole was detected for about 30 ms. The enzyme marker setting parameters are shown in Table 7.

[0071]

[0072] The detection results are shown in Table 8. Figure 5 It can be seen that chitin can trigger the active oxygen burst of the plant, and the insertion of the OsPBL8 gene does not affect this active oxygen burst. In addition, after chitin treatment, the active oxygen production level of the OsPBL8 gene overexpression plant is about 2 times higher than that of NIP.

[0073] The whole plants of OsPBL8-OE1, OsPBL8-OE6 and NIP were taken, and total RNA was extracted, and the expression of phenylalanine ammonia-lyase (PAL) gene and WRKY transcription factor 45 (WRKY45) in the tissue samples was detected by real-time fluorescent quantitative polymerase chain reaction. In the detection of the expression of phenylalanine ammonia-lyase (PAL), the primers used in reverse transcription in the real-time fluorescent quantitative polymerase chain reaction were RT-OsPAL-F and RT-OsPAL-R, and the nucleotide sequence of RT-OsPAL-F was SEQ ID No: 18, and the nucleotide sequence of RT-OsPAL-R was SEQ ID No: 19. In the detection of the expression of WRKY transcription factor 45 (WRKY45), the primers used in reverse transcription in the real-time fluorescent quantitative polymerase chain reaction were RT-OsWRKY-F and RT-OsWRKY-R, and the nucleotide sequence of RT-OsWRKY-F was SEQ ID No: 20, and the nucleotide sequence of RT-OsPAL-R was SEQ ID No: 21. The detection results are shown in Table 1. Figure 6 It can be seen that the plants overexpressing OsPBL8 gene have a significantly stronger ability to resist rice blast than the plants without inserting OsPBL8 gene.

[0074] In summary, it can be seen that overexpression of rice OsPBL8 gene can enhance the resistance of rice to Magnaporthe oryzae.

[0075] The technical features of the above-described embodiments can be combined arbitrarily, and to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not exist contradictory, it should be considered that it is within the scope of the present disclosure.

[0076] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. Application of rice OsPBL8 gene in enhancing the resistance of rice to Magnaporthe oryzae, wherein the nucleotide sequence of the OsPBL8 gene is SEQ ID No: 1, the coding sequence thereof is SEQ ID No: 2, and the amino acid sequence of the receptor-like cytoplasmic kinase encoded by the OsPBL8 gene is SEQ ID No: 3; the OsPBL8 gene sequence is transformed into an initial rice plant to form a transgenic rice with overexpression of the OsPBL8 gene.

2. Use according to claim 1, wherein The transgenic rice is formed by a method of Agrobacterium-mediated transformation of the initial rice plant by an Agrobacterium strain EHA105 loaded with the OsPBL8 gene.

3. The use according to claim 1, wherein The variety of the initial rice is Nipponbare.

4. The use according to claim 1, wherein The transgenic rice has a stronger ability to burst reactive oxygen under the induction of chitin than the initial rice.

5. The use according to claim 4, wherein the compound is ###0002### The stem base of the transgenic rice or the initial rice is soaked in a treatment liquid containing the chitin.

6. Use according to claim 5, wherein The concentration of the chitin in the treatment liquid is any value ranging from 3 μg / mL to 5 μg / mL.

7. The use according to claim 1, wherein The overexpression of the OsPBL8 gene promotes the expression of the resistance genes phenylalanine ammonia-lyase gene and WRKY transcription factor 45.

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